The neural circuitry of the dorsal horn of the spinal cord forms the basis for the mechanisms of pain and analgesia. Our lab has made significant inroads in understanding the neuronal connectivity which subserves these sensory phenomena through experiments involving multiple markers to identify interactions between neural elements. Coexistence of monoamine and peptide neurotransmitters in axons in the dorsal horn was examined using immunologically distinct multiple fluorescent markers. Coexistence of serotonin and substance P was examined initially because of the important role of serotonin in descending control of nociception. Axons which contained both serotonin and substance P were localized to all laminae of the spinal cord. The number of coexistent axons was geatest in the ventral horn and least in the the dorsal horn, especially the superficial layers which are involved in processing nociceptive information. Therefore, brain stem neurons which are the source of spinal cord coexistent serotonin and substance P axons are preferntially involved in modulation of motor rather than sensory systems. A second series of experiments used the multiple marker immunocytochemical approach in the brain stem raphe neuronal cell groups. Serotonin neurons were found to receive input from norepinephrine, enkephalin, substance P and serotonin axons. These observations suggest that serotonin neurons are modulated by other aminergic systems as well as peptidergic systems. These observations are currently being quantified in an effort to determine the frequency of these inputs. A third series of experiments relied on the production of monoclonal antibodies to produce distinctive neuronal markers in the dorsal horn. Using brain stem tissue from young rat pups as the antigen, we have produced monoclonal antibodies which identify subsets of neurons in the dorsal horn and brainstem. These markers to the cell surface's of neurons will further our ability to study the organization of dorsal horn and brain stem neural circuitry.